Pergamon 01452126(94)00161-8 Leukemia Research Vol. 19, No. 4, 291-296, 1995. pp. Copyright 0 1995 Ekevier Science Ud Printed in &at Britain. All righls reserved 0145-2126/95 $9.50 + 0.00 UPREGULATION OF p21 RAS LEVELS IN HL-60 CELLS DURING DIFFERENTIATION INDUCTION WITH DMSO, ALL-TRANS- RETINOIC ACID AND TPA Kazimierz Kuliczkowski,*t Richard L. Darley,* Allan Jacobs,m Rose Ann Padua* and Terence G. Hay* *Department of Haematology, UWMC, Heath Park, Cardiff, CF4 4XN, U.K.; TDepartment of Haematology, Pasteura 4, 50-367, Wroclaw, Poland (Received 29 June 1994. Revision accepted 24 November 1994) Abstract-The role of p21 RAS in the proliferation and differentiation of myeloid cells has been studied by analysing the changes in the level of expression of p21 RAS proteins by flow cytometry upon differentiation down the granulocytic and monocytic pathways. Differentiation resulted in upregulated p21 RAS expression despite a marked decline in the number of dividing cells. On the other hand, growth inhibition, without differentiation, resulted in a decline in expression. Cell cycle analysis showed that the increase in p21 RAS occurred throughout the cell cycle. These results suggest that p21 RAS has a role in the process of myeloid differentiation. Key words: RAS, differentiation, proliferation, cell cycle, myeloid, expression. Introduction The RAS gene family, H-, K- and N-&W code for 21 kDa proteins that have GTPase activity. Although perhaps the most widely studied oncogenes, little is known about the biology of p21 RAS proteins. The prevalence of activating RAS mutations in many types of malignancy suggests a role in cell proliferation and this is certainly the case for certain cell lines [l]. In haematological disease, RAS mutations occur at high frequency in myeloid leukaemias and at low frequency in lymphoid leukaemias [2], suggesting that p21 RAS may be particularly involved in regulating the prolifera- tion of the myeloid lineage. However, the fact that activating mutations are also found at high frequency in Abbreviations: ATR, all-trans-retinoic acid; BSA, bovine serum albumin; CD, cluster of differentiation; DMSO, dimethylsulphoxide; GM-CSF, granulocyte-macrophage col- ony stimulating factor; M-CSF, macrophage colony stimulat- ing factor; NBT, nitroblue tetrazolium; NSE, non-specific esterase; TPA, 12-0-tetradecanoylphorbol-13-acetate. Correspondence too: Dr R. L. Darley, Department of Haematology, VW&Z, Heath Park, Cardiff, CF4 4XN. (Tel: 01222 744522, Fax: 01222 744523). premalignant, non-proliferative conditions such as myelodysplasia [3] in which haematological develop- ment is disregulated, indicates that p21 RAS is also important in regulating myeloid differentiation. Studies of expression of p21 RAS proteins also support the idea that it may play a role in co-ordinating both differentiation and proliferation in different tissues; on the one hand, overexpression of p21 RAS proteins is clearly associated with some malignant conditions such as multiple myeloma [4], however, a broader survey of both neoplastic and corresponding normal tissues [5] has shown that terminally differentiated cells often express higher levels of p21 RAS than rapidly proliferating tissue. By understanding the role of RAS proteins in regulating haematopoiesis the functional significance of RAS oncogenes in leukaemia should be clarified. In this study, we have examined the expression of p21 RAS proteins during the differentiation and proliferation of the myeloid line, HL-60 [6] and correlated the distribu- tion of p21 RAS expression with cell cycle using flow cytometric analysis. They can be induced to undergo differentiation to granulocytes with dimethylsulphoxide 291